This project will result in the development of potent new inhibitors for a novel enzyme target that is specific to the metabolism of fungi and yeast: the group II self-splicing intron. This structurally complex and highly conserved ribozyme plays a key role in RNA processing and metabolic function of lower eukaryotes, but it is not present in humans and other vertebrates. Group II intron inhibitors therefore present a specific and potentially powerful new approach for combating pathogenic fungi and yeast, which represent a major public health problem for which therapeutic strategies are increasingly limited. Indeed, fungal infections are a major source of mortality and morbidity among AIDS patients, neonatal patients, and as these pathogens diversify, even among the noncompromised general population. Extensive biochemical studies have resulted in a complete enzymological framework for group II intron ribozymes, and group II intron structures in multiple stages of splicing have been thoroughly characterized through crystallography. With this groundwork in place, it is now possible to initiate a drug discovery program that is designed to identify potent small molecule inhibitors of this promising and novel target. Building on initial small molecule screening efforts (which yielded inhibitors with Ki values of 2-8 M), we will synthesize the next generation of compounds, optimizing at least two types of initial leads through iterative modification of molecular scaffolds and structure-guided SAR. Small molecule design and synthesis will be done in rapid sequence with subsequent biochemical and cell-based analyses to evaluate potency and efficacy. Our goal in Phase 1 is to identify novel group II inhibitors with Ki values of < 200 nM, to evaluate their effects on growth of pathogenic fungi and bacteria (which also contain group II introns), and to evaluate compound toxicity in mammalian cells. Completion of these focused objectives will provide a solid footing for a subsequent Phase 2 program in group II intron antifungal therapeutics.